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Ionizing radiation impacts photochemical quantum yield and oxygen evolutionactivity of Photosystem II in photosynthetic microorganismsGiuseppina Rea a; Dania Esposito a; Mario Damasso a; Agnese Serafini a; Andrea Margonelli a; CeciliaFaraloni b; Giuseppe Torzillo b; Alba Zanini c; Ivo Bertalan d; Udo Johanningmeier d; Maria T. Giardi a

a IC-CNR, Area of Research of Rome, Department of Agrofood, National Research Council, Rome b ISE-CNR, Florence c National Institute of Nuclear Physics, Turin, Italy d Martin-Luther-Universität Halle-Wittenberg, Institute of Plant Physiology, Halle, Saale, Germany

Online Publication Date: 01 January 2008

To cite this Article Rea, Giuseppina, Esposito, Dania, Damasso, Mario, Serafini, Agnese, Margonelli, Andrea, Faraloni, Cecilia, Torzillo,Giuseppe, Zanini, Alba, Bertalan, Ivo, Johanningmeier, Udo and Giardi, Maria T.(2008)'Ionizing radiation impacts photochemicalquantum yield and oxygen evolution activity of Photosystem II in photosynthetic microorganisms',International Journal of RadiationBiology,84:11,867 — 877

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Ionizing radiation impacts photochemical quantum yield and oxygenevolution activity of Photosystem II in photosynthetic microorganisms

GIUSEPPINA REA1, DANIA ESPOSITO1, MARIO DAMASSO1, AGNESE SERAFINI1,

ANDREA MARGONELLI1, CECILIA FARALONI2, GIUSEPPE TORZILLO2, ALBA ZANINI3,

IVO BERTALAN4, UDO JOHANNINGMEIER4, & MARIA T. GIARDI1

1IC-CNR, Area of Research of Rome, Department of Agrofood, National Research Council, Rome, 2ISE-CNR, Florence,3National Institute of Nuclear Physics, Turin, Italy, and 4Martin-Luther-Universitat Halle-Wittenberg, Institute of Plant

Physiology, Halle (Saale), Germany

(Received 9 January 2008; revised 4 August 2008; accepted 14 August 2008)

AbstractPurpose: Long-term space exploration requires biological life support systems capable of coping with the deleterious spaceenvironment. The use of oxygenic photosynthetic microorganisms represents an intriguing topic in this context, mainly fromthe point of view of food and O2 production. The aim of the present study was to assess the effects of space ionizing radiationexposure on the photosynthetic activity of various microorganisms.Materials and methods: Ground-based irradiation experiments were performed using fast neutrons and gamma rays onmicroorganisms maintained at various light conditions. A stratospheric balloon and a European Space Agency (ESA) flightfacility were used to deliver organisms to space at the altitude of 38 and 300 km, respectively. During the balloon flight, thefluorescence activity of the organisms was real-time monitored by means of a special biosensor.Results: The quantum yield of Photosystem II (PSII), measured directly in flight, varied among the microorganismsdepending on the light conditions. Darkness and irradiation of cells at 120 and 180 mmol m72 s71 enhanced the radiation-induced inhibition of photosynthetic activity, while exposure to weaker light irradiance of 20 and 70 mmol m72 s71 protectedthe cells against damage. Cell permanence in space reduced the photosynthetic growth while the oxygen evolution capacity ofthe cells after the flight was enhanced.Conclusions: A potential role of PSII in capturing and utilizing ionizing radiation energy is postulated.

Keywords: Space ionizing radiation, Photosystem II activity, photochemistry, oxygen evolution, survival, microorganisms

Abbreviations: D1 and D2, main proteins of Photosystem II core; F0, basic fluorescence; Fm, maximumfluorescence; Fv, variable fluorescence; Fv/Fm, maximum potential quantum yield of PSII in dark-adapted cells; PAR,photosynthetically active radiation in the range 400-750 nm; PSII, Photosystem II; Pheo7, reduced pheophytin.

Introduction

The experimental activities carried out at the Russian

Mir space station and by the USA and European

Space agencies demonstrated that space environ-

ment is stressful for living organisms, the main

problems being the presence of space radiation, the

absence of gravity, low atmospheric pressure, ex-

treme temperatures, etc. For long-duration flights

such as exploratory missions to the moon and Mars,

biological life support systems will be needed,

especially for food, nutraceutics and oxygen

production (Sonnenfeld & Miller 1993, Ohnishi

et al. 2002, Biolo et al. 2003, Dicello 2003,

Yamashita et al. 2006). Like on Earth, the biomass

production will be based on photosynthesis. Oxy-

genic photosynthetic organisms are unique in the

biosphere, since they can use light energy to split

water and evolve oxygen by means of PSII in a

process that produces storable energy-rich products

from atmospheric carbon dioxide (Barber 2006).

Therefore, with respect to future space colonization,

research on the response of the photosynthetic PSII

apparatus to space conditions is of great interest.

Correspondence: Maria Teresa Giardi, IC-CNR (National Research Council), Department of Agrofood, Area of Research of Rome, via Salaria km 29,3- 00016

Monterotondo Scalo, Rome, Italy. Tel: þ39 0690672704. Fax: þ39 0690672630. E-mail: giardi@mlib.cnr.it

Int. J. Radiat. Biol., Vol. 84, No. 11, November 2008, pp. 867–877

ISSN 0955-3002 print/ISSN 1362-3095 online � 2008 Informa Healthcare USA, Inc.

DOI: 10.1080/09553000802460149

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Space ionizing radiation is characterized by fluxes of

complex radiation of variable energy and intensity

that are very difficult to measure. It may be classified

according to its origin as: galactic cosmic radiation,

with energies between 1 and 103 GeV, comprising

protons, alpha particles and high-ionizing high

energy particles (HZE); solar energetic radiation,

from particles emitted during solar storms, mainly

composed of protons with energies that can reach

about 1 GeV; geomagnetically-trapped particle ra-

diation, comprising electrons with energies up to 7

MeV, protons with energies up to several hundreds

of MeV, and low energy heavy ions. HZE and

neutrons represent the components with the highest

relative biological effectiveness. They are particularly

dangerous since their interaction with the shielding

material of spacecraft leads to the formation of

secondary radiation such as gamma rays and

neutrons with various energies that have high

biological effectiveness (Mitaroff & Silari 2002,

Miroshnichenko 2003). HZE constitutes only 1%

of the total space ionizing radiation while neutrons

constitute 20%.

The interaction of ionizing radiation with biologi-

cal material can cause damage to DNA and proteins

modifying their structural and biochemical proper-

ties. Damage could occur by direct deposition of

energy or indirectly through the generation of

reactive oxygen species derived from water break-

down (i.e., free electrons, hydrogen free radicals, or

hydroxyl radicals). Genetic damage can also arise

from DNA repair or errors in replication of damaged

DNA, or chromosome aberration leading to cell

death and carcinogenesis (Vladimirov et al. 1992,

Benkhaled et al. 2006).

Several studies report that the absence of gravity

could affect some physiological parameters of

higher plants (Clement 2005). Microgravity is able

to modify the ultrastructure of storage reserves in

mature dry seeds and the quality of seeds produced

by space-grown Brassica rapa L. plants without

affecting reproduction (Musgrave et al. 2000,

Kuang et al. 2000, 2005). In space, the absence

of gravity particularly affects the development of

the root apparatus of higher plants; evidence of

root zone hypoxia in Brassica rapa L. grown in

microgravity has been biochemically and cyto-

chemically demonstrated and correlated to

microgravity-induced changes in fluid and gas

distribution (Stout et al. 2001). Higher plants can

partially tolerate the reduced atmospheric pressure

(or hypobaria) that, without a proper increase in

CO2 partial pressure, is responsible of a reduced

plant growth (Corey et al. 1997, Massimo & Andre

1999). Richards and colleagues analysed the con-

sequences of low pressure environments on arabi-

dopsis plants and indicated the pressure values

observed for normal plant growth (Richards et al.

2006).

Studies in unicellular organisms suggest that space

flights can cause changes in growth rate, mobility,

developmental cycle and morphological and ultra-

structural characteristics (Wang et al. 2004, Lehto

et al. 2006). The specific target of the damaging

effect on the fine structure of the photosynthetic

apparatus was determined in pea, brassica and

arabidopsis plants grown in space or during clinor-

otation. An overall decrease of photosynthetic

activities was observed. Moreover, modifications of

the chloroplast ultrastructural features were observed

mainly in the Photosystem I (PSI) and, to a lesser

extent in the PSII apparatus (Jiao et al. 2004).

A major problem for microorganisms in space

seems to be related to the presence of ionizing

radiation. Despite the numerous studies carried out

on plants and microorganisms under space condi-

tions, very little is known about the effects of space

ionizing radiation on PSII at molecular level. PSII is

the supramolecular pigment-protein complex that

catalyses the light-induced transfer of electrons from

water to plastoquinone with the oxidation of water

molecules, generating the Earth’s entire atmospheric

oxygen. PSII consists of more than 25 polypeptides,

making up the oxygen-evolving complex (OEC),

a light-harvesting chlorophyll protein complex

(LHCII) and a reaction center core composed of

two proteins, D1 and D2, which are involved in the

primary charge separation. LHCII captures light

energy and transfers it to the reaction center

chlorophyll (P680). This excitation leads to a

primary charge separation and the formation of

chlorophyll P680þ with reduced pheophytin

(Pheo7). The radical pair is stabilized when Pheo7

transfers its electron to QA, the primary quinone

acceptor bound to the D2 protein, and successively

to the secondary quinone acceptor QB. The electron

is then transferred via the cytochrome b6/f complex

and plastocyanin to PSI for NADP reduction and the

synthesis of organic compounds (Giardi & Pace

2005, Barber 2006). For the space experiments

presented here, we selected Chlorella sorokiniana,

Chlorella zofingiensis, Chlamydomonas reinhardtii,

Arthrospira platensis, Chlorococcum sp., and Monodus

subterraneus, since they represent significant stages in

the evolutionary scale of photosynthesis. Another

important reason supporting our choice was the

potential application of such organisms as a source of

nutraceuticals in space missions; the nutritional

value of these microorganisms relies in their high

content of protein, polyunsaturated fatty acids,

vitamins and carotenoids (Torzillo et al. 2003,

Walker et al. 2005).

Ground experiments using irradiation sources are

limited by the fact that it is usually possible to obtain

868 G. Rea et al.

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only single, monoenergetic radiation, often produced

in a narrow unidirectional single beam. On the

contrary in space an organism is exposed to a shower

of radiation with a wide spectrum of atomic mass and

energies between 1 MeV and 103 GeV (Miroshni-

chenko 2003). Therefore, we conducted experiments

in real-time space conditions in a stratospheric

balloon flight, at 38 km altitude, and in a Soyuz

flight at about 300 km altitude. A fluorescence-based

sensor was utilized to monitor in flight the photo-

synthetic electron transfer activity of PSII (Angeli

et al. 2001, Esposito et al. 2002, 2006).

Materials and methods

Chemicals, strains and culture conditions

All reagents were purchased from Sigma Chemical

Co., St Louis, MO, USA. Wild-type Chlorella

sorokiniana, Chlorella zofingiensis, Chlorococcum sp.,

Chlamydomonas reinhardtii (Chlorophyta), Arthrospira

platensis (Cyanobacteria), and Monodus subterraneus

(Eustigmatophyta) cells were obtained from the

culture collection at the Institute for Ecosystem

Study-National Research Council (CNR) of Flor-

ence (Tuscany, Italy). Microalgae and cyanobacteria

cultures were grown in liquid Tris/acetate/phosphate

(TAP) medium, pH 7.0 (Gorman & Levine 1965)

and Blue Green 11 (BG11) liquid medium, respec-

tively (Rippka et al. 1979), at 258C and 50 mmol

m72 s71 photon irradiance. Cells were harvested

during the exponential growth phase by centrifuga-

tion and resuspended in the same medium at a

chlorophyll (Chl) concentration of 50 mg ml71. Cell

suspensions were used for the determination of

oxygen evolution or layered on agar-medium in

plastic devices for the flight and irradiation experi-

ments. Microorganisms were subsequently exposed

to light intensities ranging from 0–180 mmol m72

s71 according to the specific experimental require-

ments.

Immunoblot analyses and densitometry

For immunoblot analyses, thylakoid membranes

were isolated from cell cultures at the exponential

growth phase, after gamma radiation exposure. Cell

cultures were harvested by centrifugation at 3,000 g

for 3 min at 48C. Pellets were diluted with sonication

buffer containing 100 mM tris(hydroxymethyl)

aminomethane-HCl (Tris-HCl) (pH 6.8), 10 mM

NaCl, 1 mM p-aminobenzamidine-2HCl, 1 mM

6-aminocaproic acid, 10 mM ethylene diamine

tetra-acetic acid (EDTA), and 100 mM phenyl-

methanesulphonylfluoride (PMSF). Cells were

disrupted by sonication for 2 min in a Branson

Sonifier Cell Disruptor 200 (Branson, Ultrasonics

Corp., Danbury, CT, USA) operated in the pulsed

mode with a 50% duty cycle and an output power

setting of 5. Unbroken cells and other large cell

fragments were removed by centrifugation at 3,000 g

for 3 min at 48C. Chlorophyll concentration was

measured upon pigment extraction in 80% acetone

after removal of cell debris by centrifugation, and by

measuring the absorbance of the solutions at 652 nm

with a Perkin Elmer Lambda BIO spectrophot-

ometer (Perkin Elmer, Monza, Lombardy, Italy)

(Geiken et al. 1998).

Samples containing equal amounts of chlorophyll

(5 mg lane71 and lower quantities) were separated on

denaturing discontinuous 12–17% polyacrylamide

gel as previously described (Geiken et al. 1998).

After electrophoresis, gels were stained with Coo-

massie brilliant blue, destained and photographed.

Alternatively, resolved proteins were electrotrans-

ferred onto a nitrocellulose filter using the Trans-

Blot Transfer Cell (Bio-Rad, Milan, Lombardy,

Italy) for 1 h at 300 mA at 48C. Filters were blocked

with high salt 16TBST (20 mM Tris-HCl [pH

8.0], 500 mM NaCl, 0.05% Tween 20), washed with

low salt 16TBST, and incubated with specific

polyclonal antibodies against the OEC, comprising

the extrinsic proteins of 33, 23, and 16 kDa and

against the D1 N-terminus, D2, CP43 and CP47

core proteins, kindly provided by Dr R. Barbato

(University of Alessandria, Piedmont, Italy). All

antibodies were diluted 1:2,000 in high salt

16TBST. For signal detection, secondary antibo-

dies conjugated to alkaline phosphatase (Promega,

Milan, Lombardy, Italy) were used at a concentra-

tion of 1:5,000 (in 16TBST) and the reaction was

visualized using 5-bromo-4-chloro-3-indoyl-phos-

phate (BCIP) and nitro blue tetrazolium (NBT)

(Promega). Proteins were quantified on filters by

scanning densitometry using a Shimadzu CS 930

densitometer (Shimadzu, Tokyo, Japan). The

amount of proteins was analysed in several dilutions

to determine the linear zone of the corresponding

standard curve.

PSII fluorescence measurements

The PSII fluorescence measurements were carried

out with an automatic multi-fluorimeter interfaced

with the cells (‘‘BioLumi’’, Biosensor S.r.l., Rome,

Lazio, Italy). The bio-device provides a simultaneous

determination in various samples of the main

chlorophyll fluorescence parameters, such as basic

fluorescence F0, maximum fluorescence Fm, variable

fluorescence Fv, and as a result, the Fv/Fm ratio, and

the area over the fluorescence curve (Papageorgiou &

Govindjee 2005). The basic fluorescence F0 is

calculated using an algorithm that determines the

line of best fit for the initial data points recorded at

PS II in space environment 869

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the onset of illumination. This line is extrapolated to

time zero in order to determine F0. The multi-

fluorimeter also recorded the external temperature

and the light intensity by the BPW34 sensor,

purchased from RS (Milan, Lombardy, Italy).

Oxygen evolution measurements

Oxygen evolution was measured at 258C by exposing

cells to saturating irradiance in a Clark-type oxygen

electrode connected to a YSI Biological Oxygen

Monitor (model 5300, Yellow Springs, Ohio, USA).

To ensure that oxygen evolution was not limited by

the carbon source available to the cells, 100 mL of 0.5

M NaHCO3, pH 7.4 was added to a 2.5 ml aliquot

of the culture prior to the oxygen evolution

measurements (Melis et al. 1999). Measurements

were performed with the O2 electrode, beginning

with the registration of dark respiration in the cell

suspension and followed by measurement of the

light-saturated rate of O2 evolution. The rate of each

process was recorded for about 5 minutes. To

compare the relative photon yield of photosynthesis

between the different samples, about the same Chl

concentration (15 mg l71) was loaded in the oxygen

electrode chamber.

Statistics

All statistical tests were performed using analysis of

variance (ANOVA). The statistical significance of

differences was evaluated by p-level. P-values have

been calculated comparing protein levels, fluores-

cence values and oxygen concentration in irradiated

samples with respect to the control.

Stratospheric balloon and space flight

Chlorella sorokiniana, Chlorella zofingiensis, Chlorococ-

cum sp., and Monodus subterraneus cultures were

layered as described above and integrated in a bio-

device carried to an average altitude of 38 km by a

200 m high balloon. The balloon, inflated with

helium, was launched from the Milo base (Trapany,

Sicily, Italy) of the Italian Space Agency. The bio-

device was recovered some hours later in Spain,

about 1400 km from the launch site. The external

box containing the microorganisms and the bio-

fluorimeter had a 1 cm-thick aluminium-polycarbo-

nate wall with a steel frame (described by Angeli

et al. 2001), able to withstand temperature changes

from7608C toþ708C. The experiments were con-

ducted at internal constant pressure of 0.8 atm. The

box was fixed to the frame of the balloon by six 4

mm-thick electrically isolated supports, in order to

keep its lower surface always in the shade. It could

also be insufflated with air to maintain a controlled

temperature. To avoid excess lighting of the micro-

organisms, the external solar light was filtered

through a black cover reducing the external illumi-

nation by 90%.

For the space flight, Chlamydomonas reinhardtii

cultures were layered on agar-TAP medium (see

above) enclosed in a sealed device. In this medium,

the algae can potentially grow photoautotrophically

as well as chemioheterotrophically in the presence of

acetate as a carbon source. Many replicates of the

same culture were inserted into the experimental

flight module. The final payload was mounted inside

the ESA facility called Biopan (www.esa.int). Biopan

is an unmanned and recoverable capsule successfully

used to deliver scientific experiments to near orbit

since 1992. The experimental module was located in

the Biopan bottom part and the temperature was

maintained at 158C. Biopan was fixed at the external

wall of a Foton spacecraft, which was launched to

250–300 km altitude. Once in orbit Biopan was

opened and algae were directly exposed to space

conditions. Algae were protected from vacuum, heat

and cold as well as from a too intense visible and

ultraviolet (UV) radiation, but they were exposed to

all other kinds of radiation without any metal cover

protection. Visible (VIS) and UV radiation protec-

tion was obtained using a combination of special

glass and quartz filters (provided by Kayser-It,

Livorno, Italy) that provided attenuation of the

external solar light between 95–99%.

The satellite, with a 7.8 km/s speed, orbited the

Earth in 90 min. During this time it was exposed to

the sun for 55 min and in darkness for 35 min.

Because of the spacecraft self-rotation, Biopan

experienced a permanent change between light and

dark during 55 min. The Biopan was opened for a

total of 351 h; at the same time the Foton spacecraft

orbited the Earth 234 times. One day before return-

ing to Earth, Biopan was closed and after nearly 250

orbits and about 379 h in flight the capsule landed in

an uninhabited area in Kazakhstan. After landing, the

experimental module was opened in sterile condi-

tions and the solid agar containing algae was

transferred to a 500 ml flask filled with 200 ml TAP

medium. The flasks were gently shaken overnight at

50 mmol m72 s71 on a rotator to completely resus-

pend the cells. From each flask 1 ml culture was

removed and plated onto a TAP solid medium to

estimate the number of surviving cells. Further sam-

pling was carried out to determine the oxygen evolution

and the growth rate during a four-day period.

Space radiation environment and radiation source

facilities

For the period of the balloon flight, an evaluation of

the radiation environment was performed in order to

870 G. Rea et al.

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determine the relative contribution to the measured

dose of the various types of ionizing radiation present

in the stratosphere. Because of the difficulty in

measuring different radiation components at the

same time over a wide energy range, both experi-

mental detection techniques and a Monte Carlo

simulation were used. An accurate neutron dose

assessment was performed considering the high

biological effectiveness of neutrons due to their

quality factors (Miroshnichenko 2003). Integral

neutron dose equivalent rates in the range of 100

keV–20 MeV were directly detected by BD 100 R

neutron bubble dosimeters. The neutron spectrum

in the 10 keV–20 MeV range was analysed by a

passive detector system based on the BDS spectro-

meter and by an unfolding technique according to

Zanini et al. (2001, 2005).

The Monte Carlo method was applied in order to

evaluate the total absorbed dose, using the GEANT3

code (GEometry ANd Tracking), that simulates the

passage of various particles through the matter in the

10 keV–10 TeV energy range (Zanini et al. 2005).

Protons (87%) were considered as a source of

primary particles, with the following distribution:

FprimaryðEp;XÞ ¼ E�gp exp �Xð1� ZÞðg�1Þ

lp

" #

where Fprimary¼ energy distribution of the primary

particles (protons) impinging on the top of the

atmosphere; Ep4 1 GeV¼ kinetic energy of primary

particles; g¼ 2.7, spectral index; Z¼ 0.5, coefficient

of elasticity; lp¼ 90 g/cm2, absorption mean free path

(for protons); and X¼ atmospheric depth (g/cm2).

A flux of protons with the mentioned energy

distribution (1074-103 GeV) interacted at the top of

the atmosphere with a flux of 0.3 protons cm72 s71

sr71. The atmosphere was simulated with 15 layers

of decreasing density; the GEANT3 code made it

possible to obtain the spectra of various secondary

particles produced by proton interaction with oxygen

(21%), nitrogen (78%), hydrogen (0.3%) and argon

(0.75%) nuclei. In particular, the neutron energy

spectra at the balloon flight altitude were evaluated

using the conversion factors from the ambient dose

equivalent (H*) to fluency taken from ICRP74

(International Commission on Radiological Protec-

tion, cf. reference ICRP, 1996), where the corre-

sponding spectra in terms of H* as a function of

neutron energy were obtained (Zanini et al. 2001,

2005).

During the 15.6 days of Foton space flight, the cell

cultures, covered by a 2 mm glass top, received a

dose of *3.4 mGy, as extrapolated from the

measurements of the active dosimeter R3D-B2

located in the Biopan facility (Dachev et al. 2005).

Information about the balloon and space flights

concerning altitude, duration and ionizing radiation

doses experienced by the organisms is summarized in

Table I.

During the space flight, the solar and absolute

radiation intensities received by the samples in

Biopan was 54.8 SCh (1 SCh¼ 4932 kJ/m26 h) as

communicated by ESA. According to the position

of the device and the measured excitation

light peaks during the rotation around the Earth,

the minimum and maximum light intensities

reaching the samples are presented in the Results

section.

Various sources of radiation were used for

experiments on the ground. A source facility is

installed at one of the secondary beam lines of the

Super Proton Synchrotron in CERN (Conseil

Europeen pour la Recherche Nucleaire). A positive

hadron beam (35% protons, 61% pions, 4% kaons)

of energy 120 GeV is secured in a copper target

which can be installed in two different positions

inside an irradiation cave. The neutron spectrum

has a second pronounced maximum at about 70

MeV which resembles the high-energy component

of the radiation field created by cosmic rays at plane

flight altitude (Mitaroff & Silari 2002). The

intensity of the primary beam is monitored by an

air-filled precision ionization chamber (PIC) at

atmospheric pressure. A PIC-count corresponds to

2.26 104 particles (error+ 10%) impinging on the

target. The energy distribution of the various

secondary particles at the various exposure locations

were obtained by means of Monte Carlo simula-

tions performed using the Fluka Code. Another

radiation source facility was the Dosimetry Division

of the Joint Research Centre (JRC) at Ispra, Varese,

Lombardy, Italy, where gamma radiation was

obtained using a 60Co source.

The details of the radiation source facilities,

energies and doses utilized for ground tests are

reported in Table II.

Table I. Details of the stratospheric and space flight experiments.

Time is given as Coordinated Universal Time (UTC) or days.

Mission

Altitude

(km) Duration

Radiation

dose rate

Stratospheric

balloon

*38 From 2 July 2001

(05:47 UTC)

0.8+ 0.3

mSv/hour

To 3 July 2001

(02:32 UTC)

Foton-M2

Space

250–300 *15.6 days

(from 31 May to

16 June 2005)

8.9+ 0.9

mGy/hour

(average

value)*

*The dosimeter used in the Foton flight calculates the absorbed

dose in Gray. At the present time, for this type of instrument, no

method is available to convert the absorbed dose into dose

equivalent.

PS II in space environment 871

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Results and discussion

PAR influence on the cell response to neutron and gamma

radiation

With a view to future space colonization it is

important to improve our knowledge of the defence

responses elicited by cosmic radiation in oxygenic

microorganisms and determine the growth condi-

tions that maximize the oxygen evolution capacity of

space-grown microalgae cultures (Lehto et al. 2006).

However, understanding the response of biological

organisms to space stress is complicated by the

multifaceted nature of space ionizing radiation

(Miroshnichenko 2003).

In this study we analyzed the behavior of green

algae and cyanobacteria in response to ionizing

radiation in both ground experiments and space

flights.

A variety of ionization radiation sources were used

to examine the effects of radiation on the photo-

chemical activities of PSII in ground experiments.

Chlamydomonas reinhardtii cells were exposed to a

total dose of 4.8 mSv fast neutrons at different PAR

as indicated in Figure 1. Cells irradiated in the dark

showed a 40% reduction in Fv/Fm ratio. In contrast,

the Fv/Fm ratio actually increased by 18% in cells

simultaneously exposed to neutron radiation and to

70 mmol m72 s71 light intensity (Figure 1). Light

intensity of 120 mmol m72 s71 and higher were

photoinhibitory, inducing a 20% decrease in Fv/Fm

values compared to the non-irradiated controls. In

the latter, the Fv/Fm values remained high over time

at all various light conditions (not shown). In

irradiated cells, oxygen evolution increased with the

increase in PAR from 20–120 mmol m72 s71 and

then declined.

Figure 1. Changes in the Fv/Fm ratio and in the oxygen evolution of Chlamydomonas reinhardtii after exposure to fast neutrons under various

light intensities. Exposure to fast neutrons was carried out at CERN: energy 0–800 MeV, dose rate 0.23 mSv h71 for a total dose 4.8 mSv.

The initial fluorescence value was Fv/Fm 0.753+0.003. The percentage of Fv/Fm modification is relative to a non-irradiated control kept

under the same light conditions. The values were obtained as the difference between the photosynthetic efficiency of irradiated and non-

irradiated samples as a percentage of the control. Oxygen evolution was measured using a Clark electrode after irradiation of 15 mg l71 Chl

in liquid culture under saturated light conditions. The experiments were repeated five times. Standard deviation (SD) is reported in the

graph. P-values�0.05 were calculated as reported in Materials and methods.

Table II. Kinds of ionizing radiation and facilities used in ground experiments.

Ionizing

radiation

quality

Ground-based

facility Radiation source Energy (MeV) Equivalent dose rate Equivalent total dose

Neutrons CERN

(Switzerland)

CERF, Super

Proton

Synchrotron

0–800 0.23 mSv/h 4.8 mSv

Gamma rays JRC Ispra

(Italy)

60Co 1.54 Sv/h 3.1 Sv

2.5 4.2 Sv/h 8.4 Sv

4.2 Sv/h 4 10 Sv

872 G. Rea et al.

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Concerning cells exposed to g-rays and treated

with doses of 3.1 Sv, no modification of the

photochemical quantum yield was observed (data

not shown). At a dose of 8.4 Sv, which is much

higher than that experienced in short-term

space flights, the photochemical quantum yield

decreased only slightly in all microorganisms, with

minor differences among the species (Table III).

The Fv/Fm values decreased slightly, but repro-

ducibly, in the following order: Arthrospira

platensis (Cyanobacteria), Chlamydomonas reinhardtii

(Chlorophyta), Monodus subterraneus (Eustigmato-

phyta), Chlorococcum sp., Chlorella sorokiniana and

Chlorella zofingiensis (Chlorophyta). In contrast, the

oxygen evolving capacity increased in all the

irradiated strains and in particular in Chlorella

(Table III).

In order to understand the effects of radiation on

the PSII apparatus at the biochemical level,

Arthrospira platensis, Chlamydomonas reinhardtii and

Chlorella sorokiniana cells were exposed to high

doses (4 10 Sv) of gamma radiation for 12 h and

the corresponding isolated thylakoids were analysed

with polyclonal antibodies against the major PSII

proteins. The results show that the exposition to

such doses causes a 40% reduction of the 33, 23

and 16 KDa OEC extrinsic protein accumulation,

compared to non-irradiated controls, in all the

analysed microorganisms. On the contrary, the

accumulation levels of the D1 and D2 protein were

slightly affected, while the content of the internal

antennae chlorophyll proteins 43 and 47 (CP43 and

CP47) appeared to remain constant, as measured

by densitometric analyses reported in Table IV.

These results are consistent with the observations

on Brassica rapa plants flown aboard the space

shuttle Columbia in 1997, where the levels of OEC

proteins in the cotyledons were found to decrease

by 32% with only slight reductions of D1, D2 and

LHCII proteins (Jiao et al. 2004).

Survival and viability of microorganisms in response to

stratospheric and space flights

In order to unravel the response of the photosyn-

thetic apparatus to real space conditions, micro-

organisms were delivered to the stratosphere at a

maximum altitude of 38 km by a balloon flight. The

algal species that in simulation studies proved to be

relatively more tolerant to ionizing radiation were

sent to space in the balloon flight under various

experimental conditions: in the light, in the dark and

in the dark partially shielded from space radiation.

The fluorescence activity was monitored by the

special automatic biodevice described in Materials

and methods.

During the balloon flight the solar activity was at

very low levels and relevant X-ray flares were not

observed. Figure 2 shows that the Eustigmatophyta

Monodus subterraneus maintained the highest photo-

chemical efficiency after the period in the strato-

sphere, while Chlorella sorokiniana showed the

highest inhibition. Dark conditions in flight particu-

larly affected the activity of Chlorella sorokiniana, but

in a less efficient way in shielded samples (Figure 2).

The passive physical dosimeters inside the box

detected a dose equivalent rate of 0.8+ 0.3 mSv

hour71 (Table I). The main radiation component

seemed to be protons (51%), while neutrons were

about 35%, gamma ray was less than 13% and HZE

less than 1%, distribution in accordance with data by

Spurny (2001).

During the Foton-M2 space mission, Chlamydo-

monas reinhardtii cells were sent to 250–300 km

altitude in a special container, where it experienced

solar radiation of different intensities using glass

attenuation filters (see Materials and methods). The

light experienced by the cells was variable depending

on the rotation of the spacecraft, but it was always in

the physiological range (2–180 micromoles photon

m72 s71). A total ionizing radiation dose of * 3.4

Table III. Changes in the Fv/Fm ratio and in the oxygen evolution in microorganisms during exposure to gamma radiation. The values were

obtained as the difference between the photosynthetic efficiency of irradiated and non-irradiated samples as a percentage of the control.

Oxygen evolution was measured by a Clark electrode at saturating light intensity. Exposure to gamma ray at JRC-ISPRA (Exposure time

20 min; total dose 8.4 Sv) with illumination at 70 mmol m72s71. Data (mean values+SD, n¼5) are expressed as percentages relative to

non-irradiated controls. P-values �0.05 were calculated as reported in Materials and methods.

Organisms Average of cell size Control Fv/Fm

% Fv/Fm % 02

relative to non-irradiated

control

Arthrospira platensis 10 mm 0.759+ 0.003 96+2.4 101+ 2

Chlamydomonas reinhardtii 10 mm 0.750+ 0.003 96+2.1 115+ 2

Monodus subterraneus 9.0 mm 0.760+ 0.001 95+2.0 102+ 2

Chlorococcum sp. 4.0 mm 0.781+ 0.004 94+2.2 107+ 5

Chlorella zofingiensis 3.7 mm 0.749+ 0.002 93+1.9 122+ 7

Chlorella sorokiniana 3.7 mm 0.777+ 0.003 90+2.1 115+ 6

PS II in space environment 873

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mGy was measured during the flight. After the flight,

all algae retained the green and damp phenotype and

also the medium consistency and colour were not

affected. Following the re-suspension procedures,

the algae were used to estimate the cell surviving

capability, the growth rate and the oxygen evolution

capacity (Table V).

The cells plated on TAP solid medium formed

colonies after 14 days. Surviving cells were found

with a percentage of 0.001% and 0.01% in algae

exposed to 1% VIS-UV radiation (range 2–10 mmol

m72 s71) and 5% VIS radiation (range 30–80 mmol

m72 s71), respectively; on the contrary no survivors

were observed with 10% VIS radiation (range 90–

180 mmol m72 s71). The two survival cultures were

further analysed comparing their growth rate in liquid

media to those obtained in the on ground control

culture. The comparison showed that an increase of

light intensity decreases the algae percentage growth

rate to about 80%. Interestingly, although the growth

rate was lowered, the survival culture improved

oxygen production up to 22%, exceeding the rate

found in on ground controls (Table V).

The first observation is that, in space, the effect of

ionizing radiation is enhanced compared to that

observed in on ground facilities with a single beam of

radiation; in fact in the stratospheric flight, PSII

activity was affected even at the low doses of 20 mSv

(see Figure 2). The sensitivity of PSII to ionizing

radiation seems to be reasonable since it works as an

Table IV. PSII protein accumulation in green algae and

cyanobacteria following exposure to high doses of gamma

radiation. Cultures of the different microorganisms at a

exponential growth phase were exposed to a 60Co source for

12 h, receiving a total dose410 Sv. Thylakoid membranes were

isolated from harvested cell cultures and their membrane

proteins fractionated by SDS-PAGE (12–17% gradients; Geiken

et al. 1998). Gels were loaded on an equal chlorophylls basis

(5 mg lane71) and the proteins were detected by BCIP and

NBT colorimetric assay. Proteins were quantified on filters by

scanning densitometriy and the amount of proteins was analysed

in several dilutions to determine the linear zone of the

corresponding standard curve. D1 and D2: heterodimeric

protein of the PSII reaction centre; 33, 23 and 16 KDa: OEC

extrinsic proteins of the PSII; CP43 and CP47: internal

antennae proteins of the PSII reaction centre. Data (mean

values+SD, n¼ 5) are expressed as percentage relative to non-

irradiated controls. P-values �0.05 were calculated as reported

in Materials and methods.

Strain PSII proteins

% protein relative

to non irradiated

control

Arthrospira platensis D1 85+11

D2 90+7

OEC (33-23-16) 64+4

CP43þCP47 98+5

Chamydomonas

reinhardtii

D1 82+12

D2 87+7

OEC (33-23-16) 62+6

CP43þCP47 95+4

Chlorella

sorokiniana

D1 81+13

D2 83+8

OEC (33-23-16) 59+6

CP43þCP47 94+4

Figure 2. Changes in the Fv/Fm ratio in microalgal samples after a 10-hour balloon flight in the stratosphere at 38 km altitude. The cells

received external solar light filtered through a black cover reducing the illumination by 90%. During the flight, a light intensity range of

15–40 mmol m72s71 was measured. Average temperatures were 258+ 58C and a total dose of 20 mSv was determined. Shield: samples were

partially shielded from radiation by a 5 mm thick cadmium cover layer. Double samples were analysed in triplicate measurements. Standard

deviations (SD) are reported in the graph.

874 G. Rea et al.

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electron transport chain extremely sensitive to the

presence of free radicals (Jansen et al. 1999, Booij-

James et al. 2000).

During the stratospheric flight, the main distur-

bance to the PSII apparatus complex seemed to be

caused by ionizing radiation, since the effect on its

activity was similar to that registered in ground

experiments.

In the experiment in space, Chlamydomonas

reinhardtii was only protected by 2 mm thick glass

filters during exposure to the space environment.

The new discovery is that photosynthetically active

cells can, in principal, survive under such extreme

conditions. Once again, it was observed that the

effect of space stress on algae survival varies

depending on the light conditions to which they

were exposed during the flight. Surprisingly, despite

the low survival of the organisms, the flight experi-

ence caused a stimulation of PSII oxygen evolution

of the cells, as also registered in ground experiments

with neutrons and gamma rays (Figure 1 and

Table IV). This contradictory phenomenon has been

already observed in other organisms e.g., Thalassio-

sira weissglogii and Monodus subterraneus under stress

(Torzillo et al. 2003, and Giardi et al. unpublished

results). In these species it was found that a decrease

in the population of functional PSII reaction centres

of up to 50% could be compensated by means of an

increase in the rate of electron transport rate through

the resting functional PSII reaction centres. The

increased oxygen evolution activity in cells exposed

in space may be the result of modification of

thylakoid stacking due the presence of new charged

ions and to the formation of hydroxyl radicals,

triggered by the ionizing radiation. It is known that

oxygen evolution can originate from hydroxyl radi-

cals that in normal conditions are produced by

mitochondria respiration (Pospısil et al. 2004).

It was interesting to observe that the consequence

of space stress and gamma exposure on the micro-

organisms seems to be inversely correlated to cell

dimensions. Monodus subterraneus was significantly

less affected than Chlorella sorokiniana and Chlorella

zofingiensis. Indeed, among the organisms tested,

Chlorella (Chlorophyta), which showed the highest

sensitivity, is the smallest in size (average cell

diameter: 3.7 mm). On the other hand, Monodus

subterraneus (Eustigmatophyta), which showed the

least inhibition, has the largest diameter (more than

9 mm) (Figure 2). Similar results were obtained

following gamma ray exposure which, in addition to

Monodus subterraneus, indicates that the largest

microorganisms Chlamydomonas reinhardtii and

Arthrospira platensis are the less sensitive microorgan-

isms (Table III). It seems that large cell cross-

sections, with their content of lipids, antioxidants

and enzymes, can partially shield internal structures.

However, other possible factors could account for

the higher sensitivity observed in green algae as

opposed to cyanobacteria such as, for instance, the

content of photoprotective pigments. In a screening

of microalgal species exposed to UV-B, damage was

correlated to cell dimensions, in accordance with our

findings on ionizing radiation damage; the lower the

cell diameter, the higher the damage recorded

(Xiong et al. 1996).

The OEC of PSII seems to be particularly

affected by ionizing radiation resulting in a

stimulation at the lowest doses of mSv and in a

reduction of the OEC proteins at the highest doses

over 10 Sv. Interestingly PAR plays a very

important synergistic role in the response of PSII

to ionizing radiation: both in the dark and under

relatively intense illumination, the damaging effect

of space radiation increased, while the effect of

ionizing radiation was negligible under weak light

(Figure 1). Perhaps this synergistic effect is due to

a phenomenon similar to photoinhibition; both

ionizing radiation and high light intensities are

known to cause the formation of free radicals that,

in addition, could damage the photosynthetic

apparatus. A similar synergism has been previously

observed by Jansen and colleagues (1996) in the

aquatic higher plant Spirodela, where a simulta-

neous exposure to PAR and ultraviolet-B radiation

determined an increase of the D1-D2 heterodimer

protein degradation (Jansen et al. 1996). This

behaviour suggests that the light-dependent D1

protein turn-over may play a role in the PSII

resistance to ionizing radiation stress. This assump-

tion is in accordance with previous observations

that D1 protein is a main target of high light

radiation damage in PSII and is supported by the

fact that recovery of D1 content in the dark is

known to be precluded (Mattoo et al. 1999).

Table V. Survival and oxygen evolution activity of Chlamydomonas

reinhardtii after a space flight. After 1 week re-suspension, oxygen

evolution was measured by a Clark electrode at saturating light

intensity in post-flight re-cultured cells at 15 mg l71 Chl

concentration. Growth rate was followed over four days and

reported as percentage compared to on-ground control cells. The

measurements were repeated three times. The SD is indicated.

Range of light

intensity

(mmol m72s71)

% Post-flight

survivors

% Oxygen

evolution

% Growth

rate

Maximum and

minimum

peak experienced

during flight

Values relative

to initial

layered cells

Relative to on

ground control

2–10 0.001 103+ 3 82+ 4

30–80 0.01 122+ 6 80+ 4

90–180 nr – –

PS II in space environment 875

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The synergetic effect of ionizing radiation and light

on the photosynthetic apparatus must be considered

in relation to the survival of photosynthetic organ-

isms in space since bio-regenerative life-support

systems can be severely disturbed. All the analysed

microorganisms – Chlorella sorokiniana, Chlorella

zofingiensis, Chlamydomonas reinhardtii, Arthrospira

platensis, Chlorococcum sp., and Monodus subterraneus

– on account of their high nutritional value and

human health-promoting activity might be suitable as

bio-regenerative life supporting systems in space

(Torzillo et al. 2003, Walker et al. 2005). In

particular, in our experiments Arthrospira platensis

and Chlamydomonas reinhardtii seem to maintain the

highest photosynthetic efficiency in flight experi-

ments. Moreover, several data in the literature

address the potential of the multicellular, filamen-

tous cyanobacterium Arthrospira platensis as a life

supporting system since it shows tolerance to heat,

high light intensity and alkaline environment (Lehto

et al. 2006).

In addition, radioresistant strains of Chlorella and

Chlamydomonas have been previously isolated and

characterized, revealing the presence of all the basic

DNA repair pathways following radiation exposure.

The connection between the radioresistance and the

adaptive repair processes is linked to other cellular

functions like the regulation of the cell cycle, which is

strictly correlated to developmental processes, man-

datory for every bioregenerative supporting system in

space (Boreham & Mitchel 1993, Chankova et al.

2005, Vlbek et al. 2008).

The ability to adjust in the presence of ionizing

radiation existed in the early stages of the Earth’s

history, prior to the establishment of photosynthesis.

The creation of an oxygenic atmosphere, with its

ozone layer, protected Earth against space radiation

and led to the creation of microorganisms incapable

of adapting to space conditions. In accordance with

this knowledge, our experiments have demonstrated

that ionizing radiation can modify the photochemical

properties of PSII and the oxygen-evolving apparatus

activity. These observations raise intriguing ques-

tions about a potential role of PSII in ionizing

radiation energy capture and utilization.

Acknowledgements

This work was supported by ASI Biotechnology and

Medicine with a grant to Maria Teresa Giardi and by

BMBF/DLR with the grant No. 50WB0420 to Udo

Johanningmeier. The flight experiment was sup-

ported by ESA.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are respon-

sible for the content and writing of the paper.

References

Angeli G, Ragni P, Esposito D, Giardi P, Pompili ML,

Moscardelli R, Giardi, MT. 2001. A device to study the effect

of space radiation on photosynthetic organisms. Physica

Medica 17 (Suppl. 1):267–268.

Barber J. 2006. Photosystem II: An enzyme of global significance.

Biochemical Society Transaction 34:619–631.

Benkhaled L, Barrios L, Mestres M, Caballin MR, Ribas M,

Barquinero JF. 2006. Analysis of gamma-rays induced

chromosome aberrations: A fingerprint evaluation with

a combination of pan-centromeric and pan-telomeric

probes. International Journal of Radiation Biology 82:869–

875.

Biolo G, Heer M, Narici M, Strollo F. 2003. Microgravity as a

model of ageing. Current Opinion in Clinical Nutrition and

Metabolic Care 6:3–7.

Booij-James IS, Dube SK, Jansen MA, Edelman M, Mattoo AK.

2000. Ultraviolet-B radiation impacts light-mediated turnover

of the photosystem II reaction center heterodimer in Arabi-

dopsis mutants altered in phenolic metabolism. Plant Physiol-

ogy 124(3):1275–1284.

Boreham DR, Mitchel RE. 1993. DNA repair in Chlamydomonas

reinhardtii induced by heat shock and gamma radiation.

Radiation Research 135:365–371.

Chankova GS, Matos JA, Simoes F, Bryant PE. 2005. Adaptive

response of a new radioresistant strain of Chlamydomonas

reinhardtii and correlation with increased DNA double-

strandbreak rejoining. International Journal of Radiation

Biology 81(7):509–514.

Clement G, editor. 2005. Space medicineNew York: Springer.

ISBN 1402015984.

Corey KA, Barta DJ, Henninger DL. 1997. Photosynthesis

and respiration of a wheat stand at reduced atmospheric

pressure and reduced oxygen. Advances in Space Research

20:1869–1877.

Dachev T, Dimitrov P, Tomov B, Matviichuk Y, Bankov N,

Hader DP. 2005. Observation of the Earth radiation

environment by R3D-B2 instrument on Foton M2 satellite.

Proceedings of the 11th International Scientific Conference

on Solar-Terrestrial Influences, Sofia (Bulgaria); pp 171–174.

Dicello JF. 2003. The impact of the new biology on radiation risks

in space. Health Physiology 85:94–1027.

Esposito D, Faraloni C, Fasolo F, Margonelli A, Torzillo G,

Zanini A, Giardi MT. 2006. Biodevices for space research. In:

Giardi MT, Piletska E, editors. Biotechnological applications

of photosynthetic proteins: Biochips, biosensors and biode-

vices, Landes Bioscience; pp 192–208.

Esposito D, Pace E, Margonelli A, Rizzuto M, Giardi P, Giardi

MT. 2002. A biodosemeter that utilises isolated enzymes to

detect of ionising radiation. Radiation Protection Dosimetry

99:303–305.

Geiken B, Masojidek J, Rizzuto M, Pompili ML, Giardi MT.

1998. Incorporation of [35S] methionine in higher plants

reveals that stimulation of the D1 reaction centre II protein

turnover accompanies tolerance to heavy metal stress. Plant

Cell and Environment 21:1265–1273.

Giardi MT, Pace E. 2005. Photosynthetic proteins for

technological applications. Trends in Biotechnology 23:257–

263.

Gorman DS, Levine RP. 1965. Cytochrome f and plastocyanin:

Their sequence in the photosynthetic electron transport chain

of Chlamydomonas reinhardi. Proceedings of the National

Academy of Sciences of the USA 54:1665–1669.

ICRP (International Commission on Radiological Protection).

1996. Conversion coefficient for use in radiological protection

against external radiation. ICRP Publication 74, Annals of

ICRP, 26, n 3/4.

876 G. Rea et al.

Downloaded By: [HEAL-Link Consortium] At: 09:07 8 June 2009

Jansen MA, Gaba V, Greenberg BM, Mattoo AK, Edelman M.

1996. Low threshold levels of ultraviolet-B in a background of

photosynthetically active radiation trigger rapid degradation

of the D2 protein of Photosystem II. The Plant Journal

9(5):693–699.

Jansen MA, Mattoo AK, Edelman M. 1999. D1-D2 protein

degradation in the chloroplast. Complex light saturation

kinetics. European Journal of Biochemistry 260(2):527–532.

Jiao S, Hilaire E, Paulsen AQ, Guikema JA. 2004. Brassica rapa

plants adapted to microgravity with reduced Photosystem I

and its photochemical activity. Physiologia Plantarum 122:

281–290.

Kuang A, Popova A, McClure G, Musgrave ME. 2005. Dynamics

of storage reserve during Brassica rapa L. pollen and seed

development in microgravity. International Journal of Plant

Sciences 166:85–96.

Kuang A, Xiao Y, McClure G, Musgrave ME. 2000. Influence of

microgravity on ultrastructure and storage reserves in seeds of

Brassica rapa L. Annals of Botany 85:851–859.

Lehto KM, Lehto HJ, Kanervo EA. 2006. Suitability of different

photosynthetic organisms for an extraterrestrial biological life

support system. Research in Microbiology 157:69–76.

Massimo D, Andre M. 1999. Growth of wheat under one tenth of

the atmospheric pressure. Advances in Space Research

24:293–296.

Mattoo A, Giardi MT, Raskind A, Edelman M. 1999. Dynamic

metabolism of photosystem II reaction center proteins and

pigments. A review. Physiologia Plantarum 107:454–461.

Melis A, Neidhardt J, Benemann JR. 1999. Dunaliella salina

(Chlorophyta) with small chlorophyll antenna sizes exhibit

higher photosynthetic productivities and photon use efficien-

cies than normally pigmented cells. Journal of Applied

Phycology 10:515–525.

Miroshnichenko LI. 2003. Radiation hazard in space, Vol. 297.

Dordrecht, The Netherlands: Astrophysics and Space Science

Library.

Mitaroff A, Silari M. 2002. The CERN-EU high-energy reference

field (CERF) facility for dosimetry at commercial flight

altitudes and in space. Radiation Protection Dosimetry

102:7–22.

Musgrave ME, Kuang A, Xiao Y, Stout SC, Bingham GE, Briarty

LG, Levenskikh MA, Sychev VN, Podolski IG. 2000. Gravity

independence of seed-to-seed cycling in Brassica rapa. Planta

210:400–406.

Ohnishi T, Takahashi A, Ohnishi K. 2002. Studies about space

radiation promote new fields in radiation biology. Journal of

Radiation Research 43:7–12.

Papageorgiou GC, Govindjee. 2005. Chlorophyll a fluorescence:

A signature of photosynthesis. 1st ed., The Netherlands:

Springer Verlag.

Pospısil P, Arato A, Krieger-Liszkay A, Rutherford AW. 2004.

Hydroxyl radical generation by photosystem II. Biochemistry

43:6783–6792.

Richards JT, Corey KA, Paul AL, Ferl RJ, Wheeler RM,

Schuerger AC. 2006. Exposure of Arabidopsis thaliana to

hypobaric environments: implications for low-pressure bior-

egenerative life support systems for human exploration mis-

sions and terraforming to Mars. Astrobiology 6:851–866.

Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY.

1979. Generic assignments, strain histories and properties of

pure cultures of cyanobacteria. Journal of General Microbiol-

ogy 111:1–61.

Sonnenfeld G, Miller ES. 1993. Space flight and humoral

and cellular immunity of animals. Physiologist 36(Suppl. 1):

S68–70.

Spurny F. 2001. Radiation doses at high altitudes and during

space flights. Radiation Physics and Chemistry 61:301–307.

Stout SC, Porterfield DM, Briaty LG, Kuang A, Musgrave ME.

2001. Evidence of root zone hypoxia in Brassica rapa L. grown

in microgravity. International Journal of Plant Sciences

162:249–255.

Torzillo G, Goksan T, Faraloni C, Kopecky J, Masojıdek J. 2003.

Interplay between photochemical activities and pigment

composition in an outdoor culture of Haematococcus pluvialis

during the shift from the green to red stage. Journal of Applied

Phycology 15:127–136.

Xiong F, Lederer F, Lukavsky J, Nedbal L. 1996. Screening of

freshwater algae (Chlorophyta, Chromophyta) for ultraviolet-B

sensitivity of the photosynthetic apparatus. Journal of Plant

Physiology 148:42–48.

Vladimirov VG, Belokhvostov AS, Sherlina SS, Vasilyeva IN,

Voskresensky AM. 1992. Extracellular DNA level in the blood

of irradiated rats. International Journal of Radiation Biology

62:667–671.

Vlbek D, Sevcovicova A, Sviezena B, Galova E, Miadokova E.

2008. Chlamydomonas reinhardtii: A convenient model system

for the study of DNA repair in photoautotrophic eukaryotes.

Current Genetics 53:1–22.

Walker TL, Purton S, Becker DK, Collet C. 2005. Microalgae as

bioreactors. Plant and Cell 24:629–641.

Wang GH, Li GB, Li DH, Liu YD, Song LR, Tong GH, Liu XM

and Cheng ET. 2004. Real-time studies on microalgae under

microgravity. Acta Astronautica 55:131–137.

Yamashita M, Ishikawa Y, Kitaya Y, Goto E, Arai M, Hashimoto

H, Tomita-Yokotani K, Hirafuji M, Omori K, Shiraishi A,

Tani A, Toki K, Yokota H, Fujita O. 2006. An overview of

challenges in modeling heat and mass transfer for living on

Mars. Annals of New York Academy Sciences 1077:232–243.

Zanini A, Ongaro C, Manfredotti C, Tommasino L, and Miranda

Losa P. 2001. Neutron spectrometry at various altitudes in

atmosphere by passive detector technique. Il Nuovo cimento

della Societa italiana di fisica C 24C:471–784.

Zanini A, Storini M, Visca L, Durisi EAM, Perosino M, Borla O,

Saavedra O. 2005. Neutron spectrometry at high mountain

observatories. Journal of Atmospheric and Solar-Terrestrial

Physics 67:755–762.

PS II in space environment 877

Downloaded By: [HEAL-Link Consortium] At: 09:07 8 June 2009